Methods for controlling the proliferation of cells

ABSTRACT

The invention shows that 12-oxo-phytodienoic acid, coronatine, 6-azido-1-oxo-indanoyl isoleucine and related compounds arrest growth and induce cell death of exponentially proliferating cancer cells and malignant B-cell lymphoma cell lines in a dose dependant manner. It appears that the compounds of the present invention are not toxic to cancer cells but rather irreversibly effect cell growth, differentiation and the interaction between the cancer cell and the extracellular matrix.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 60/461,426, filed Apr. 10, 2003, thedisclosure of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the treatment ofdisease states that result from uncontrolled cell proliferation, andmore specifically to the treatment of cancerous conditions usingcompounds that activate higher plant defense systems.

[0004] 2. Discussion of the Related Art

[0005] Over the past twenty years only a few drugs isolated from higherplants have yielded clinical agents, the outstanding examples beingvinblastine and vincristine from the Madagascan periwinkle, Catharanthusroseus, etoposide, the semi-synthetic lignam, from May-apple Podophyllumpeltatum and the diterpenoid taxol, commonly referred to as paclitaxel,from the Pacific yew, Taxus brevifolia. Of these agents, paclitaxel isthe most exciting, having received approval by the Food and DrugAdministration for the treatment of refractory ovarian cancer. Since theisolation of octadecanoid-derived hormones, there has been a concertedeffort by investigators to study other therapeutic applications ofoctadecanoid-derived hormones their derivatives and compounds that mimicthe same.

[0006] The oxidative metabolism of polyunsaturated fatty acids in plantsgives rise to a group of biologically active compositions known asoctadecanoid-derived hormones or oxylipins. Jasmonic acid (“JA”) and12-oxo-phytodienoic acid (“12-oxo-PDA”) are the two well known activeoxylipins in this class, although there is mounting evidence that otheroctadecanoids are also active. In plants, the oxylipins are involved ina variety of regulatory functions, including growth regulation, woundresponse, resistance to pathogenic attacks, senescence, fruit ripening,chilling tolerance and tuberization. For example, the oxylipins play asignificant and essential role in signal transduction processes betweenexternal stimuli (e.g., wounding or pathogenic attack) and the cellularresponses thereto (e.g., activation of defense genes such as proteinaseinhibitors).

[0007] In plants, 12-oxo-PDA (I) is generated by the sequential actionof allene oxide synthase and allene oxide cyclase on linoleic acid thathas been oxygenated with lipoxygenase.

[0008] JA (II) can then be synthesized from 12-oxo-PDA by reductionfollowed by beta-oxidation.

[0009] Other compounds similar in structure to 12-oxo-PDA demonstratephytotoxic effects. Coronatine (III), for example, is a highly activephytotoxin produced by several pathovars of Pseudomonas syrinage (e.g.,tomato, glycinea).

[0010] Coronatine, much like 12-oxo-PDA, can activate the stresssignaling processes in plants, which, for example, result in volatileemission or tendril coiling. Indeed, coronatine shares both structuraland biological properties with 12-oxo-PDA, thus suggesting thatcoronatine will have the same or similar effects as 12-oxo-PDA if usedin a mammalian system.

[0011] Similarly, synthetic 6-azido-1-oxo-indanoyl isoleucine (IV)resembles 12-oxo-PDA and coronatine.

[0012] As with coronatine, 6-azido-1-oxo-indanoyl isoleucine (IV) caninduce volatile emissions and/or tendril coiling. This compound wassynthesized to contain an azido photoaffinity label in order todetermine the site of action of coronatine.

[0013] The use of JA and its derivatives to affect plant growth and cropimprovements has been described. Application of jasmonates can have awide range of effects on many plant species, ranging from the inhibitionof plant development to the promotion of plant processes. For example,it has been shown that use of jasmonic acid ester and giberellin cansynergistically enhance plant growth and development. Alternatively, ithas been demonstrated that use of particular jasmonates can inhibitsprouting and darkening in tubers after picking. In addition, it hasbeen shown that use of methyl jasmonate helps repel insects.

[0014] The levels of JA, 12-oxo-PDA and other intermediates producedduring oxylipin synthesis vary considerably among various plant species.These variations in concentration suggest that the relative and absoluteconcentrations of different oxylipins may provide flexibility withinthis multifunctional chemical signaling system. Specifically, thesefindings indicate that there are at least two structurally well-definedprocessing systems (e.g., receptors and/or binding proteins) that areselective for either of JA or 12-oxo-PDA.

[0015] Some protein and signal transduction components involved in cellcycle regulation and apoptosis have been highly conserved among plantsand animals during evolution. For example, many plant products (such astaxol) are widely used as growth regulators and apoptotic agents inmammalian systems—especially in the field of cancer treatment. There isstill a need, therefore, to identify drug candidates that haveactivities that are equivalent to or greater than those of knownantiproliferative agents.

SUMMARY OF THE INVENTION

[0016] The invention relates generally to the treatment of diseases anddisorders that result from uncontrolled cell proliferation. Morespecifically, this invention provides methods of controlling cellproliferation, comprising administering to a subject in need thereof atleast one compound having the Formula V:

[0017] wherein

[0018] —is an optional double bond;

[0019] A¹ and A² are independently H, Z_(m)-OR⁶, oxo, halo, Z_(m)-CN,Z_(m)-NO₂, azido, Z_(m)-NR⁶R⁷, Z_(m)-COOR⁶, Z_(m)-CONR⁶R⁷,Z_(m)-C(═O)R⁶, Z_(m)-OC(═O)R⁶, alkyl, allyl, alkenyl, alkynyl,heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy,thiol, thioalkyl, Z_(m)-cycloalkyl wherein said cycloalkyl is saturatedor partially unsaturated, Z_(m)-heterocycloalkyl wherein saidheterocycloalkyl is saturated or partially unsaturated, or Z_(m)-Ar¹,wherein said alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl,heteroalkenyl, heteroalkynyl, heteroalkoxy, Z_(m)-cycloalkyl,Z_(m)-heterocycloalkyl, and Z_(m)-Ar¹ may be substituted orunsubstituted;

[0020] A³ and A⁴ are independently alkyl, allyl, alkenyl, alkynyl,heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy,Z_(m)-cycloalkyl wherein said cycloalkyl is saturated or partiallyunsaturated, Z_(m)-heterocycloalkyl wherein said heterocycloalkyl issaturated or partially unsaturated, Z_(m)-Ar, Z_(m)-O—R⁶, Z_(m)-SR⁶,Z_(m)-NR⁶R⁷, Z_(m)-C(═O)R⁶, Z_(m)-OC(═O)R⁶, Z_(m)-C(═O)OR⁶,Z_(m)-(C═O)NR⁶R⁷, or Z_(m)-NHC(═O)R⁶, wherein said alkyl, allyl,alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl,heteroalkynyl, heteroalkoxy, Z_(m)-cycloalkyl, Z_(m)-heterocycloalkyl,and Z_(m)-Ar may be substituted or unsubstituted and wherein at leastone of A³ or A⁴ is at least three atoms in length;

[0021] or A³ and A⁴ together with the atoms to which they are bothattached form a substituted or unsubstituted saturated or partiallyunsaturated ring or a substituted or unsubstituted aromatic ring havingat least five atoms, wherein one or more of the atoms is optionally aheteroatom;

[0022] R⁶ and R⁷ are independently H, Z_(m)-OR⁶, alkyl, allyl, alkenyl,alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl,heteroalkoxy, Z_(m)-cycloalkyl wherein said cycloalkyl is saturated orpartially unsaturated, Z_(m)-heterocycloalkyl wherein saidheterocycloalkyl is saturated or partially unsaturated, or Z_(m)-Ar,wherein said alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl,heteroalkenyl, heteroalkynyl, heteroalkoxy, Z_(m)-cycloalkyl,Z_(m)-heterocycloalkyl, and Z_(m)-Ar may be substituted orunsubstituted;

[0023] X is OR⁶, oxo, heteroalkoxy, O-glucosyl, thiol, thioalkyl, NR⁶R⁷,halo, CN, NO₂, or azido;

[0024] Ar is aryl or heteroaryl;

[0025] Z is CH₂; and

[0026] m is an integer between 0 and 10.

[0027] In one embodiment, the method comprises administering a compoundsuch as 12-oxo-PDA (I), coronatine (III), 6-azido-1-oxo-indanoylisoleucine (IV), and related compounds to regulate cell growth and deathin proliferating cells such as human malignant neoplastic cells.

[0028] The present invention further provides a pharmaceuticalcomposition for controlling proliferative cells in a subject, comprisinga therapeutically effective amount of a compound having the formula:

[0029] and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings, which are included to provide afurther understanding of the invention and are incorporated in andconstitute a part of this specification, illustrate embodiments of theinvention and together with the description serve to explain theprinciples of the invention.

[0031] In the drawings:

[0032]FIG. 1 is a magnification of breast cancer cells treated with12-oxo-PDA versus a control sample measured at three and eight-dayintervals with arrowed indications of accumulated lipid droplets in thecytoplasm;

[0033]FIG. 2 is a magnification of lung cancer cells treated with12-oxo-PDA versus a control sample measured at three and eight-dayintervals with arrowed indications of accumulated lipid droplets in thecytoplasm;

[0034]FIG. 3 is a magnification of lung cancer cells treated withvarious concentrations of 12-oxo-PDA versus a control sample measuredafter eight days of treatment;

[0035]FIG. 4 is a magnification of human malignant diffuse large B-celllymphoma cells showing morphological changes measured at two andeight-day intervals resulting from treatment with 12-oxo-PDA versus acontrol sample;

[0036]FIG. 5 is a chart illustrating the effect on cell viability formetastatic human breast cancer cells, lung cancer cells and malignanthuman B-cell lymphoma cells measured after eight days of treatment with12-oxo-PDA;

[0037]FIG. 6 is a chart illustrating the effects on growth and number ofmetastatic human breast cancer cells, lung cancer cells and malignanthuman B-cell lymphoma cells in the presence or absence of 12-oxo-PDA;

[0038]FIG. 7 is a chart comparing the effects on cell growth and numberof human breast cancer cells in the presence of 12-oxo-PDA versusjasmonic acid;

[0039]FIG. 8 is a chart comparing the effects on cell growth and numberof CRL-2632 lymphoma cells in the presence of 12-oxo-PDA versus jasmonicacid

[0040]FIG. 9 is a gel electrophoresis demonstrating that 12-oxo-PDAinduces inhibition of cyclin D1 expression and Rb phosphorylation;

[0041]FIG. 10 is an amplification of the regulatory effects on cyclin D1mRNA following treatment with ethanol and 12-oxo-PDA;

[0042]FIG. 11 is an amplification of the regulatory effects on p27 mRNAfollowing treatment with ethanol and 12-oxo-PDA;

[0043]FIG. 12 is an amplification of the regulatory effects on C-FOSmRNA following treatment with ethanol and 12-oxo-PDA;

[0044]FIG. 13 is a gel electrophoresis illustrating the effects of12-oxo-PDA on apoptosis and inhibits expression of the MCL-1 protein;

[0045]FIG. 14 is a graph displaying the growth ratio of CRL-2632lymphoma cell numbers in the presence or absence of synthetic coronatine(CRNT) over ethanol (control) plates.

[0046]FIG. 15 is a gel electrophoresis illustrating the effects of12-oxo-PDA and coronatine on the expression of the MCL-1 protein; and

[0047]FIG. 16 is a gel electrophoresis illustrating the effects of12-oxo-PDA on beta-tubulin polymerization.

DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention is directed towards methods of controllingproliferative cells in a subject by administering a therapeuticallyeffective amount of a compound having the Formula V:

[0049] wherein:

[0050] —is an optional double bond;

[0051] A¹ and A² are independently H, Z_(m)-OR⁶, oxo, halo, Z_(m)-CN,Z_(m)-NO₂, azido, Z_(m)-NR⁶R⁷, Z_(m)-C(═O)OR⁶, Z_(m)-C(═O)NR⁶R⁷,Z_(m)-C(═O)R⁶, Z_(m)-OC(═O)R⁶, alkyl, allyl, alkenyl, alkynyl,heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy,thiol, thioalkyl, Z_(m)-cycloalkyl wherein said cycloalkyl is saturatedor partially unsaturated, Z_(m)-heterocycloalkyl wherein saidheterocycloalkyl is saturated or partially unsaturated, or Z_(m)-Ar,wherein said alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl,heteroalkenyl, heteroalkynyl, heteroalkoxy, Z_(m)-cycloalkyl,Z_(m)-heterocycloalkyl, and Z_(m),-Ar¹ may be substituted orunsubstituted;

[0052] A³ and A⁴ are independently alkyl, allyl, alkenyl, alkynyl,heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy,Z_(m)-cycloalkyl wherein said cycloalkyl is saturated or partiallyunsaturated, Z_(m)-heterocycloalkyl wherein said heterocycloalkyl issaturated or partially unsaturated, Z_(m)-Ar, Z_(m)-O—R⁶, Z_(m)-SR⁶,Z_(m)-NR⁶R⁷, Z_(m)-C(═O)R⁶, Z_(m)-OC(═O)R⁶, Z_(m)-C(═O)OR⁶,Z_(m)-(C═O)NR⁶R⁷, or Z_(m)-NHC(═O)R⁶, wherein said alkyl, allyl,alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl,heteroalkynyl, heteroalkoxy, Z_(m)-cycloalkyl, Z_(m)-heterocycloalkyl,and Z_(m)-Ar may be substituted or unsubstituted and wherein at leastone of A³ or A⁴ is at least three atoms in length;

[0053] or A³ and A⁴ together with the atoms to which they are bothattached form a substituted or unsubstituted saturated or partiallyunsaturated ring or a substituted or unsubstituted aromatic ring havingat least five atoms, wherein one or more of the atoms is optionally aheteroatom;

[0054] R⁶ and R⁷ are independently H, Z_(m)-OR⁶, alkyl, allyl, alkenyl,alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl,heteroalkoxy, Z_(m)-cycloalkyl wherein said cycloalkyl is saturated orpartially unsaturated, Z_(m)-heterocycloalkyl wherein saidheterocycloalkyl is saturated or partially unsaturated, or Z_(m)-Ar,wherein said alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl,heteroalkenyl, heteroalkynyl, heteroalkoxy, Z_(m)-cycloalkyl,Z_(m)-heterocycloalkyl, and Z_(m)-Ar may be substituted orunsubstituted;

[0055] X is OR⁶, oxo, heteroalkoxy, O-glucosyl, thiol, thioalkyl, NR⁶R⁷,halo, CN, NO₂, or azido;

[0056] Ar is aryl or heteroaryl;

[0057] Z is CH₂; and

[0058] m is an integer between 0 and 10.

[0059] The compounds of this invention can be used to treat or preventproliferative diseases or disorders in mammals, including but notlimited to humans, in which cells grow and increase in number rapidly.For example, the lymphoproliferative disorders are diseases in whichthere is malignant growth of lymphoid cells and of cells from thereticuloendothelial system (which take up and sequester inertparticles). As another example, the myeloproliferative disorders aremalignancies of certain bone marrow cells including those that give riseto the red blood cells, the granulocytes (types of white blood cells),and the platelets (crucial to blood clotting).

[0060] Proliferative diseases which may be treated or prevented include,but are not limited to, non-small-cell lung cancer, colon, CNS,melanoma, ovarian, renal, prostate and breast cancers, lymphoma,leukemias including acute myelogenous leukemia and chronic myelogenousleukemia, metastatic melanoma, Kaposi's sarcoma, and multiple myeloma.

[0061] The term “alkyl” as used herein refers to a saturated linear orbranched-chain monovalent hydrocarbon radical of one to twelve carbonatoms, wherein the alkyl radical may be optionally substitutedindependently with one or more substituents described below. Examples ofalkyl groups include, but are not limited to, methyl, ethyl, n-propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl,tert-pentyl, hexyl, isohexyl, and the like.

[0062] The term “alkenyl” refers to linear or branched-chain monovalenthydrocarbon radical of two to twelve carbon atoms, containing at leastone double bond, e.g., ethenyl, propenyl, and the like, wherein thealkenyl radical may be optionally substituted independently with one ormore substituents described herein, and includes radicals having “cis”and “trans” orientations, or alternatively, “E” and “Z” orientations.

[0063] The term “alkynyl” refers to a linear or branched monovalenthydrocarbon radical of two to twelve carbon atoms containing at leastone triple bond. Examples include, but are not limited to, ethynyl,propynyl, and the like, wherein the alkynyl radical may be optionallysubstituted independently with one or more substituents describedherein.

[0064] The term “allyl” refers to a radical having the formula RC═CHCHR,wherein R is alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,aryl, heteroaryl, or any substituent as defined herein, wherein theallyl may be optionally substituted independently with one or moresubstituents described herein.

[0065] The term “cycloalkyl” refers to saturated or partiallyunsaturated cyclic hydrocarbon radical having from three to twelvecarbon atoms, wherein the cycloalkyl may be optionally substitutedindependently with one or more substituents described herein. The term“cycloalkyl” further includes bicyclic and tricyclic cycloalkylstructures, wherein the bicyclic and tricyclic structures may include asaturated or partially unsaturated cycloalkyl fused to a saturated orpartially unsaturated cycloalkyl or heterocycloalkyl ring or an aryl orheteroaryl ring. Examples of cycloalkyl groups include, but are notlimited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, and the like.

[0066] The term “heteroalkyl” refers to saturated linear orbranched-chain monovalent hydrocarbon radical of one to twelve carbonatoms, wherein at least one of the carbon atoms is replaced with aheteroatom selected from N, O, or S, and wherein the radical may be acarbon radical or heteroatom radical. The heteroalkyl radical may beoptionally substituted independently with one or more substituentsdescribed herein. The term “heteroalkyl” encompasses alkoxy andheteroalkoxy radicals.

[0067] The term “heterocycloalkyl” refers to a saturated or partiallyunsaturated cyclic radical of 3 to 8 ring atoms in which at least onering atom is a heteroatom selected from nitrogen, oxygen and sulfur, theremaining ring atoms being C where one or more ring atoms may beoptionally substituted independently with one or more substituentdescribed below. The radical may be a carbon radical or heteroatomradical. “Heterocycloalkyl” also includes radicals where heterocycleradicals are fused with aromatic or heteroaromatic rings. Examples ofheterocycloalkyl rings include, but are not limited to, pyrrolidine,piperidine, piperazine, tetrahydropyranyl, morpholine, thiomorpholine,homopiperazine, phthalimide, and derivatives thereof.

[0068] The term “heteroalkenyl” refers to linear or branched-chainmonovalent hydrocarbon radical of two to twelve carbon atoms, containingat least one double bond, e.g., ethenyl, propenyl, and the like, whereinat least one of the carbon atoms is replaced with a heteroatom selectedfrom N, O, or S, and wherein the radical may be a carbon radical orheteroatom radical. The heteroalkenyl radical may be optionallysubstituted independently with one or more substituents describedherein, and includes radicals having “cis” and “trans” orientations, oralternatively, “E” and “Z” orientations.

[0069] The term “heteroalkynyl” refers to a linear or branchedmonovalent hydrocarbon radical of two to twelve carbon atoms containingat least one triple bond. Examples include, but are not limited to,ethynyl, propynyl, and the like, wherein at least one of the carbonatoms is replaced with a heteroatom selected from N, O, or S, andwherein the radical may be a carbon radical or heteroatom radical. Theheteroalkynyl radical may be optionally substituted independently withone or more substituents described herein.

[0070] The term “heteroallyl” refers to radicals having the formulaRC═CHCHR, wherein R is alkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, or any substituent as definedherein, wherein at least one of the carbon atoms is replaced with aheteroatom selected from N, O, or S, and wherein the radical may be acarbon radical or heteroatom radical. The heteroallyl may be optionallysubstituted independently with one or more substituents describedherein.

[0071] “Aryl” means a monovalent aromatic hydrocarbon monocyclic radicalof 6 to 10 ring atoms or a polycyclic aromatic hydrocarbon, optionallysubstituted independently with one or more substituents describedherein. More specifically the term aryl includes, but is not limited to,phenyl, 1-naphthyl, 2-naphthyl, and derivatives thereof. The term “aryl”further includes heteroaryl rings.

[0072] “Heteroaryl” means a monovalent monocyclic aromatic radical of 5to 10 ring atoms or a polycyclic aromatic radical, containing one ormore ring heteroatoms selected from N, O, or S, the remaining ring atomsbeing C. The aromatic radical is optionally substituted independentlywith one or more substituents described herein. More specifically theterm heteroaryl includes, but is not limited to, furyl, thienyl,pyrrolyl, pyridyl, pyrazolyl, pyrimidinyl, imidazolyl, pyrazinyl,indolyl, thiophen-2-yl, quinolyl, benzopyranyl, thiazolyl, andderivatives thereof.

[0073] The term “halo” includes fluoro, chloro, bromo or iodo.

[0074] As used herein, the terms “treatment” and “treating” refer to anymethodology, regimen, or protocol for affecting or altering a mammaliancondition or disease, including, without limitation, (1) preventing adisease or condition from occurring in a subject that may be predisposedto the disease or condition but has not yet been diagnosed with thedisease or condition, (2) inhibiting a disease or condition (i.e.,arresting the development of the disease or condition), and (3)relieving the disease or condition (i.e., causing a regression of thedisease or condition).

[0075] In general, the various moieties or functional groups of thecompounds of Formulas I and III-IX may be optionally substituted by oneor more substituents. Examples of substituents suitable for purposes ofthis invention include, but are not limited to, halo, alkyl, allyl,alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl,heteroalkynyl, heteroalkoxy, Z_(y)-cycloalkyl, Z_(y)-heterocycloalkyl,Z_(y)-OR⁸, Z_(y)-NO₂, Z_(y)-CN, Z_(y)-CO₂R⁸, Z_(y)-(C═O)R⁸,Z_(y)-O(C═O)R⁸, Z_(y)-O-alkyl, Z_(y)-OAr, Z_(y)-SH, Z_(y)-SR⁸,Z_(y)-SOR⁸, Z_(y)-SO₂R⁸, Z_(y)-S—Ar, Z_(y)-SOAr, Z_(y)-SO₂Ar, aryl,heteroaryl, Z_(y)-Ar, Z_(y)-(C═O)NR⁹R¹⁰, Z_(y)-NR⁹R¹⁰, Z_(y)-NR(C═O)R,Z_(y)-SO₂NR⁸R⁹, PO₃H₂, and SO₃H₂, where:

[0076] Z is (CH₂),

[0077] y is zero or 1,

[0078] R⁸, R⁹, and R¹⁰ are independently alkyl, allyl, alkenyl, alkynyl,heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, alkoxy,heteroalkoxy, Z_(y)-cycloalkyl, or Z_(y)-heterocycloalkyl, and

[0079] Ar is aryl or heteroaryl,

[0080] wherein said alkyl, allyl, alkenyl, alkynyl, heteroalkyl,heteroallyl, heteroalkenyl, heteroalkynyl, alkoxy, heteroalkoxy,Z_(y)-cycloalkyl, Z_(y)-heterocycloalkyl, Ar, R⁸, R⁹, and R¹⁰ may befurther substituted or unsubstituted.

[0081] In one embodiment, the method comprises administering a compoundhaving Formula V as defined above, wherein A³ and A⁴ are independently

[0082] where

[0083] n is 3, 4, 5, 6, 7, 8, 9, or 10;

[0084] D₁, D₂ and D₃ are independently H, Z_(m)-OR⁶, O-glucosyl,heteroalkoxy, thiol, thioalkyl, NR⁷R⁸, halo, CN, NO₂, or azido;

[0085] D₄ is H, Z_(m)-OR⁶, O-glucosyl, imino, halo, Z_(m)-CN, Z_(m)-NO₂,azido, Z_(m)-C(═O)H, Z_(m)-NR⁶R⁷, Z_(m)-COOR⁶, Z_(m)-CONR⁶R⁷,Z_(m)-C(═O)R⁶, Z_(m)-OC(═O)R⁶, alkyl, allyl, alkenyl, alkynyl,heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy,thiol, thioalkyl, Z_(m)-cycloalkyl wherein said cycloalkyl is saturatedor partially unsaturated, Z_(m)-heterocycloalkyl wherein saidheterocycloalkyl is saturated or partially unsaturated, or Z_(m)-Ar,wherein said alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl,heteroalkenyl, heteroalkynyl, heteroalkoxy, Z_(m)-cycloalkyl,Z_(m)-heterocycloalkyl, and Z_(m)-Ar may be substituted orunsubstituted,

[0086] or D₄ and X, or D₄ and D₃ together form a lactone;

[0087] R⁷ and R⁸ are independently H, Z_(m)-OR⁶, alkyl, allyl, alkenyl,alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl,heteroalkoxy, Z_(m)-cycloalkyl wherein said cycloalkyl is saturated orpartially unsaturated, Z_(m)-heterocycloalkyl wherein saidheterocycloalkyl is saturated or partially unsaturated, or Z_(m)-Ar,wherein said alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl,heteroalkenyl, heteroalkynyl, heteroalkoxy, Z_(m)-cycloalkyl,Z_(m)-heterocycloalkyl, and Z_(m)-Ar may be substituted orunsubstituted; and

[0088] Z_(m), Ar, R⁶ and R⁷ are defined as above.

[0089] In another embodiment, the method comprises administering acompound having Formula V as defined above, wherein A³ and A⁴ areindependently

[0090] Another aspect of this invention provides a method of controllingproliferative cells in a subject, comprising administering a compoundhaving the Formula VI:

[0091] Wherein R¹ is

[0092] R², R³, R⁴ and R⁵ are independently H, Z_(m)-OR⁶, alkyl, allyl,alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl,heteroalkynyl, heteroalkoxy, Z_(m)-NR⁶R⁷, Z_(m)-COOR⁶, Z_(m)-CONR⁶R⁷,Z_(m)-C(═O)R⁶, Z_(m)-OC(═O)R⁶, Z_(m)-cycloalkyl wherein said cycloalkylis saturated or partially unsaturated, Z_(m)-heterocycloalkyl whereinsaid heterocycloalkyl is saturated or partially unsaturated, orZ_(m)-Ar, wherein said alkyl, allyl, alkenyl, alkynyl, heteroalkyl,heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy,Z_(m)-cycloalkyl, Z_(m)-heterocycloalkyl, and Z_(m)-Ar may besubstituted or unsubstituted,

[0093] or R³ and R⁴ together with the atoms to which they are bothattached form a saturated or partially unsaturated ring, wherein saidsaturated ring or partially unsaturated ring may be substituted orunsubstituted; and

[0094] Y¹, Y², and Y³ are independently H, Z_(m)-OR⁶, alkyl, allyl,alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl,heteroalkynyl, heteroalkoxy, Z_(m)NR⁶R⁷, Z_(m)-COOR⁶, Z_(m)-CONR⁶R⁷,Z_(m)-C(═O)R⁶, Z_(m)-OC(═O)R⁶, Z_(m)-cycloalkyl wherein said cycloalkylis saturated or partially unsaturated, Z_(m)-heterocycloalkyl whereinsaid heterocycloalkyl is saturated or partially unsaturated, orZ_(m)-Ar, wherein said alkyl, allyl, alkenyl, alkynyl, heteroalkyl,heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy,Z_(m)-cycloalkyl, Z_(n)-heterocycloalkyl, and Z_(m)-Ar may besubstituted or unsubstituted;

[0095] and wherein A¹, A², X, Z_(m), Ar, R⁶ and R⁷ defined as above.

[0096] In one embodiment, R¹ is a substituted or unsubstituted naturalamino acid, including, but not limited to alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, or valine; or an unnatural amino acid,including, but not limited to 4-hydroxyproline, hydroxylysine, demosine,isodemosine, 3-methylhistidine, norvaline, beta-alanine,gamma-aminobutyric acid, cirtulline, homocysteine, homoserine, omithineand methionine sulfone.

[0097] In one embodiment, the method uses a compound having the FormulaVII or VIII:

[0098] where A¹, A², Y¹, Y², Y³, and R¹ are defined as above. In onespecific embodiment, the method uses a compound having the Formula IX:

[0099] where Y² is defined as above. In a specific embodiment, Y² isazido.

[0100] Another aspect of this invention provides a method of controllingproliferative cells in a subject, comprising administering a compoundhaving the Formula I

[0101] Yet another aspect of this invention provides a method ofcontrolling proliferative cells in a subject, comprising administering acompound having the Formula III

[0102] The compounds of this invention may possess one or moreasymmetric centers; such compounds can therefore be produced asindividual (R)— or (S)— stereoisomers or as mixtures thereof. Unlessindicated otherwise, the description or naming of a particular compoundin the specification and claims is intended to include both individualenantiomers and mixtures, racemic or otherwise, thereof. Accordingly,this invention also includes racemates and resolved enantiomers, anddiastereomers compounds of the Formulas I and III-IX. The methods forthe determination of stereochemistry and the separation of stereoisomersare well known in the art (see discussion in Chapter 4 of “AdvancedOrganic Chemistry”, 4th edition J. March, John Wiley and Sons, New York,1992).

[0103] In order to use a compound of the Formula I or III-IX, or apharmaceutically acceptable salt or in vivo cleavable prodrug thereof,for the therapeutic treatment (including prophylactic treatment) ofmammals including humans, it is normally formulated in accordance withstandard pharmaceutical practice as a pharmaceutical composition.According to this aspect of the invention there is provided apharmaceutical composition that comprises a compound of the Formula I orIII-IX, or a pharmaceutically acceptable salt or in vivo cleavableprodrug thereof, as defined hereinbefore in association with apharmaceutically acceptable diluent or carrier.

[0104] The compositions of the invention may be in a form suitable fororal use (for example as tablets, lozenges, hard or soft capsules,aqueous or oily suspensions, emulsions, dispersible powders or granules,syrups or elixirs), for topical use (for example as creams, ointments,gels, or aqueous or oily solutions or suspensions), for administrationby inhalation (for example as a finely divided powder or a liquidaerosol), for administration by insufflation (for example as a finelydivided powder) or for parenteral administration (for example as asterile aqueous or oily solution for intravenous, subcutaneous, orintramuscular dosing or as a suppository for rectal dosing). Forexample, compositions intended for oral use may contain, for example,one or more coloring, sweetening, flavoring and/or preservative agents.

[0105] Suitable pharmaceutically-acceptable excipients for a tabletformulation include, for example, inert diluents such as lactose, sodiumcarbonate, calcium phosphate or calcium carbonate, granulating anddisintegrating agents such as corn starch or algenic acid; bindingagents such as starch; lubricating agents such as magnesium stearate,stearic acid or talc; preservative agents such as ethyl or propylp-hydroxybenzoate, and anti-oxidants, such as ascorbic acid. Tabletformulations may be uncoated or coated either to modify theirdisintegration and the subsequent absorption of the active ingredientwithin the gastrointestinal tract, or to improve their stability and/orappearance, in either case, using conventional coating agents andprocedures well known in the art.

[0106] Compositions for oral use may be in the form of hard gelatincapsules in which the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin, oras soft gelatin capsules in which the active ingredient is mixed withwater or an oil such as peanut oil, liquid paraffin, or olive oil.

[0107] Aqueous suspensions generally contain the active ingredient infinely powdered form together with one or more suspending agents, suchas sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone,gum tragacanth and gum acacia; dispersing or wetting agents such aslecithin or condensation products of an alkylene oxide with fatty acids(for example polyoxethylene stearate), or condensation products ofethylene oxide with long chain aliphatic alcohols, for exampleheptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives (such as ethyl orpropyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid),coloring agents, flavoring agents, and/or sweetening agents (such assucrose, saccharine or aspartame).

[0108] Oily suspensions may be formulated by suspending the activeingredient in a vegetable oil (such as arachis oil, olive oil, sesameoil or coconut oil) or in a mineral oil (such as liquid paraffin). Theoily suspensions may also contain a thickening agent such as beeswax,hard paraffin or cetyl alcohol. Sweetening agents such as those set outabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

[0109] Dispersible powders and granules suitable for preparation of anaqueous suspension by the addition of water generally contain the activeingredient together with a dispersing or wetting agent, suspending agentand one or more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients such as sweetening, flavoring and coloring agents,may also be present.

[0110] The pharmaceutical compositions of the invention may also be inthe form of oil-in-water emulsions. The oily phase may be a vegetableoil, such as olive oil or arachis oil, or a mineral oil, such as forexample liquid paraffin or a mixture of any of these. Suitableemulsifying agents may be, for example, naturally-occurring gums such asgum acacia or gum tragacanth, naturally-occurring phosphatides such assoya bean, lecithin, an esters or partial esters derived from fattyacids and hexitol anhydrides (for example sorbitan monooleate) andcondensation products of the said partial esters with ethylene oxidesuch as polyoxyethylene sorbitan monooleate. The emulsions may alsocontain sweetening, flavoring and preservative agents.

[0111] Syrups and elixirs may be formulated with sweetening agents suchas glycerol, propylene glycol, sorbitol, aspartame or sucrose, and mayalso contain a demulcent, preservative, flavoring and/or coloring agent.

[0112] The pharmaceutical compositions may also be in the form of asterile injectable aqueous or oily suspension, which may be formulatedaccording to known procedures using one or more of the appropriatedispersing or wetting agents and suspending agents, which have beenmentioned above. A sterile injectable preparation may also be a sterileinjectable solution or suspension in a non-toxic parenterally-acceptablediluent or solvent, for example a solution in 1,3-butanediol.

[0113] Suppository formulations may be prepared by mixing the activeingredient with a suitable non-irritating excipient which is solid atordinary temperatures but liquid at the rectal temperature and willtherefore melt in the rectum to release the drug. Suitable excipientsinclude, for example, cocoa butter and polyethylene glycols.

[0114] Topical formulations, such as creams, ointments, gels and aqueousor oily solutions or suspensions, may generally be obtained byformulating an active ingredient with a conventional, topicallyacceptable, vehicle or diluent using conventional procedures well knownin the art.

[0115] Compositions for administration by insufflation may be in theform of a finely divided powder containing particles of average diameterof, for example, 30 μm or much less, the powder itself comprising eitheractive ingredient alone or diluted with one or more physiologicallyacceptable carriers such as lactose. The powder for insufflation is thenconveniently retained in a capsule containing, for example, 1 to 50 mgof active ingredient for use with a turbo-inhaler device, such as isused for insufflation of the known agent sodium cromoglycate.

[0116] Compositions for administration by inhalation may be in the formof a conventional pressurized aerosol arranged to dispense the activeingredient either as an aerosol containing finely divided solid orliquid droplets. Conventional aerosol propellants such as volatilefluorinated hydrocarbons or hydrocarbons may be used and the aerosoldevice is conveniently arranged to dispense a metered quantity of activeingredient.

[0117] For further information on formulations, see Chapter 25.2 inVolume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairmanof Editorial Board), Pergamon Press 1990, which is specificallyincorporated herein by reference.

[0118] The amount of a compound of this invention that is combined withone or more excipients to produce a single dosage form will necessarilyvary depending upon the host treated and the particular route ofadministration. For example, a formulation intended for oraladministration to humans will may contain, for example, from 0.5 mg to 2g of active agent compounded with an appropriate and convenient amountof excipients which may vary from about 5 to about 98 percent by weightof the total composition. Dosage unit forms will generally contain about1 mg to about 500 mg of an active ingredient. For further information onroutes of administration and dosage regimes, see Chapter 25.3 in Volume5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman ofEditorial Board), Pergamon Press 1990, which is specificallyincorporated herein by reference.

[0119] The size of the dose for therapeutic or prophylactic purposes ofa compound of Formula I or III-IX will naturally vary according to thenature and severity of the conditions, the age and sex of the animal orpatient and the route of administration, according to well knownprinciples of medicine. For example, the method may comprise at leastone of an hourly administration, a daily administration, a weeklyadministration, or a monthly administration of one or more compositionsdescribed herein.

[0120] In addition to compounds of the Formula I and III-IX, theinvention also includes solvates, pharmaceutically acceptable prodrugs,pharmaceutically active metabolites, and pharmaceutically acceptablesalts of such compounds.

[0121] The term “solvate” refers to an aggregate of a molecule with oneor more solvent molecules.

[0122] A “pharmaceutically acceptable prodrug” is a compound that may beconverted under physiological conditions or by solvolysis to thespecified compound or to a pharmaceutically acceptable salt of suchcompound.

[0123] A “pharmaceutically active metabolite” is a pharmacologicallyactive product produced through metabolism in the body of a specifiedcompound or salt thereof. Metabolites of a compound may be identifiedusing routine techniques known in the art and their activitiesdetermined using tests such as those described herein.

[0124] Prodrugs and active metabolites of a compound may be identifiedusing routine techniques known in the art. Various forms of prodrugs areknown in the art. For examples of such prodrug derivatives, see, forexample, a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985)and Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder, etal. (Academic Press, 1985); b) A Textbook of Drug Design andDevelopment, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5“Design and Application of Prodrugs”, by H. Bundgaard p. 113-191 (1991);c) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992); d) H.Bundgaard, et al., Journal of Pharmaceutical Sciences, 77:285 (1988);and e) N. Kakeya, et al., Chem. Pharm. Bull., 32:692 (1984), each ofwhich is specifically incorporated herein by reference.

[0125] A “pharmaceutically acceptable salt” is a salt that retains thebiological effectiveness of the free acids and bases of the specifiedcompound and that is not biologically or otherwise undesirable. Acompound of the invention may possess a sufficiently acidic, asufficiently basic, or both functional groups, and accordingly reactwith any of a number of inorganic or organic bases, and inorganic andorganic acids, to form a pharmaceutically acceptable sale. Examples ofpharmaceutically acceptable salts include those salts prepared byreaction of the compounds of the present invention with a mineral ororganic acid or an inorganic base, such salts including sulfates,pyrosulfates, bisulfates, sulfites, bisulfites, phosphates,monohydrogenphosphates, dihydrogenphosphates, metaphosphates,pyrophosphates, chlorides, bromides, iodides, acetates, propionates,decanoates, caprylates, acrylates, formates, isobutyrates, caproates,heptanoates, propiolates, oxalates, malonates, succinates, suberates,sebacates, fumarates, maleates, butyn-1,4-dioates, hexyne-1,6-dioates,benzoates, chlorobenzoates, methylbenzoates, dinitromenzoates,hydroxybenzoates, methoxybenzoates, phthalates, sulfonates,xylenesulfonates, pheylacetates, phenylpropionates, phenylbutyrates,citrates, lactates, γ-hydroxybutyrates, glycollates, tartrates,methanesulfonates, propanesulfonates, naphthalene-1-sulfonates,naphthalene-2-sulfonates, and mandelates.

[0126] If the inventive compound is a base, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, or with an organic acid, such as aceticacid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonicacid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, apyranosidyl acid, such as glucuronic acid or galacturonic acid, analphahydroxy acid, such as citric acid or tartaric acid, an amino acid,such as aspartic acid or glutamic acid, an aromatic acid, such asbenzoic acid or cinnamic acid, a sulfonic acid, such asp-toluenesulfonic acid or ethanesulfonic acid, or the like.

[0127] If the inventive compound is an acid, the desiredpharmaceutically acceptable salt may be prepared by any suitable method,for example, treatment of the free acid with an inorganic or organicbase, such as an amine (primary, secondary or tertiary), an alkali metalhydroxide or alkaline earth metal hydroxide, or the like. Illustrativeexamples of suitable salts include, but are not limited to, organicsalts derived from amino acids, such as glycine and arginine, ammonia,primary, secondary, and tertiary amines, and cyclic amines, such aspiperidine, morpholine and piperazine, and inorganic salts derived fromsodium, calcium, potassium, magnesium, manganese, iron, copper, zinc,aluminum and lithium.

[0128] According to the present invention, suitable methods ofadministering the therapeutic composition of the present invention to apatient include any route of in vivo administration that is suitable fordelivering the composition into a patient. The preferred routes ofadministration will be apparent to those of skill in the art, dependingon the type of condition to be prevented or treated, and/or the targetcell population. Preferred methods of in vivo administration include,but are not limited to, intravenous administration, intraperitonealadministration, intramuscular administration, intranodal administration,intracoronary administration, intraarterial administration (e.g., into acarotid artery), subcutaneous administration, transdermal delivery,intratracheal administration, intraarticular administration,intraventricular administration, inhalation (e.g., aerosol),intracranial, intraspinal, intraocular, intranasal, oral, bronchial,rectal, topical, vaginal, urethral, pulmonary administration,impregnation of a catheter, and direct injection into a tissue.

[0129] As described in further detail below and illustrated in FIGS.1-14, the present invention demonstrates that 12-oxo-PDA (I) arrestscell growth and induces cell death of actively and exponentiallyproliferating high-grade metastatic human breast cancer cells (T-47D),lung cancer cells (CRL-5985) and malignant human B-cell lymphoma cells(CRL-2632) in a dose dependant manner. Further, the inventiondemonstrates that at similar concentrations, jasmonic acid fails to beas effective.

[0130] It was also observed that exposure of the cancer cells to12-oxo-PDA resulted in significant morphological changes whereby thecancer cells ceased three-dimensional growth patterns and became morediffuse and single layered. Additionally, 12-oxo-PDA resulted in lipidaccumulation in the cytoplasm of the cancer cells as shown by Oil Red 0staining. Importantly, the data suggests that 12-oxo-PDA (and therelated compounds) is not toxic to the cancer cells. Rather, it appearsthat 12-oxo-PDA (and the related compounds) irreversibly interferes withthe cellular mechanisms involved in cell growth, differentiation and theinteraction between the cells with the extracellular matrix. Indeed,removal of 12-oxo-PDA from the culture mediums did not restore cellgrowth.

[0131] Treatment with 12-oxo-PDA also resulted in inhibition of cyclinD1 expression and inhibition of the hyperphosphorylation ofretinoblastoma tumor suppressor protein (“Rb”) in T-47D metastatic humanbreast cancer cells.

[0132] Accordingly, one aspect of the invention is the use of 12-oxo-PDAand the related compounds disclosed herein as part of a method fortreating cancer and other conditions characterized by uncontrolled orotherwise excessive cell proliferation.

[0133] The invention is further illustrated by the following non-limitedexamples. All scientific and technical terms have the meanings asunderstood by one with ordinary skill in the art. The specific exampleswhich follow illustrate the methods in which the compositions of thepresent invention may be used and are not to be construed as limitingthe invention in sphere or scope. The methods may be adapted tovariation in order to produce compositions embraced by this inventionbut not specifically disclosed. Further, variations of the methods toproduce the same compositions in somewhat different fashion will beevident to one skilled in the art.

EXAMPLES

[0134] In the following examples, all referenced cell lines werepurchased from ATCC. T47D cells were maintained in Dulbecco's ModifiedEagle medium (DMEM) (purchased from Life Technologies, Inc.)supplemented with 10% fetal calf serum (purchased from LifeTechnologies, Inc.), insulin (5 μg/ml), and antibiotics. CRL-5985,CRL-2632 and Raji cells were maintained in RPMI-1640 medium supplementedwith 10% fetal calf serum (purchased from Life Technologies, Inc.) andantibiotics. Western blot analysis was performed by using phospho-serine807/811-Rb, Rb, cyclin D1, cyclin D3, cdk-2, cdk-4, bcl-2, phospho-p38,bax, bak, PARP (cell signaling technology), MCL-1 (purchased fromBiosource International) and beta-tubulin antibodies (purchased fromBecton-Dickenson). Proteins were separated using an 8-10%SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred toImmobilon-P membranes and immunoblotted with antibodies using the ECLdetection system.

[0135] For real-time RT-PCR experiments T-47D cells were treated withethanol (control) or with 25 uM 12-OPDA for 48 hours. Total RNA wasisolated using TRIzol reagent (Invitrogen, Carlsbad, Calif.) followed bypurification on RNeasy columns (Qiagen, Palo Alto, Calif.). Forquantitative real-time RT-PCR analyses, total RNA from each sample wasreverse transcribed with MuLV reverse transcriptase (Applied Biosystems,Foster City, Calif.) and random hexamer primers. The SYBR Green PCR Kit(Applied Biosystems) was used for quantitative real-time RT-PCRanalysis. The primers for human ubuquitin, cyclin D1, p27 and c-fos weredesigned using Primer Express software (Applied Biosystems). 12-oxo-PDAwas purchased from Larodon Fine Chemicals AB, Sweden. Jasmonic acid waspurchased from Sigma-Aldrich Co. The indanoyl amino acid conjugate ofcoronatine (i.e., synthetic 6-azido-1-oxo-indanoyl isoleucine) wasobtained from Dr. Wilhelm Boland (Max Planck Institute, Germany).

Example 1 Method for treating breast cancer cells with 12-oxo-PDA (I)

[0136] T-47D human breast ductal adenocarcinoma cells were treated with12-oxo-PDA and measured at three and eight-day intervals. As can be seenin FIG. 1, there is a significant decrease in cell growth andaccumulation in the cells treated with 12-oxo-PDA compared to theuntreated control sample.

Example 2 Method for Treating Lung Cancer Cells with 12-oxo-PDA (I)

[0137] CRL-5985 human lung adenocarcinoma cells were treated with12-oxo-PDA and measured at three and eight-day intervals. FIG. 2, whichshows a 200× magnification of the treated cells, demonstrates that incomparison to a control sample there is a significant decrease in cellgrowth and morphological changes in the cells treated with 12-oxo-PDA,whereby the cancer cells ceased three-dimensional growth patterns andbecame more diffuse and single layered.

Example 3 Method of Treating CRL-5985 Human Lung Adenocarcinoma with12-oxo-PDA (I)

[0138] Lung cancer cells were treated with concentrations of 12-oxo-PDAranging from 15 μM to 50 μM (versus an untreated control sample). Aftereight days of treatment at the various concentrations, a 200×magnification of the cells (FIG. 3) revealed a decrease in cell growthproportional to the concentration of 12-oxo-PDA supplied to the cells(i.e., treatment with higher concentrations of 12-oxo-PDA resulted in agreater decrease in cell growth).

Example 4 Method for Treating CRL-2632 Human Malignant Diffuse LargeB-cell Lymphoma Cells with 12-oxo-PDA (I)

[0139]FIG. 4 is a 200× magnification of human malignant diffuse largeB-cell lymphoma cells showing morphological changes at two and eight-dayintervals resulting from treatment with 12-oxo-PDA versus a controlsample.

Example 5 Effect of 12-oxo-PDA (I) on Cell Viability and Cell Growth

[0140] Cell lines CRL-2632 (B-cell lymphoma cells), CRL-5985 (human lungadenocarcinoma cells) and T-47D (human breast ductal adenocarcinomacells) were exposed to 12-OPDA over an eight-day period. Cell viabilityand cell growth were measured versus controls. As shown in FIG. 5,12-oxo-PDA had a significant inhibitory effect on cell viability, asdetermined by trypan blue exclusion assay. FIG. 5 illustrates that12-oxo-PDA was particularly effective at affecting the viability of thetreated CRL-2632 cells.

[0141] As demonstrated in FIG. 6, it was observed that the CRL-2632cells had approximately a ten-fold decrease in total cell numberfollowing treatment with 12-oxo-PDA (versus untreated samples over thesame period of time). Similarly, the T-47D cells exhibited anapproximate three-fold decrease in cell number while the CRL-5985 cellsdemonstrated a five-fold decrease after treatment with 12-oxo-PDA(versus untreated samples over the same period of time).

Example 6 12-Oxo-PDA (I) Versus Jasmonic Acid (II) on Cell Growth ofBreast Cancer Cells

[0142] The effects on cell growth and number for T-47D human breastcancer cells and CRL-2632 human malignant diffuse large B-cell lymphomacells following treatment with 12-oxo-PDA and jasmonic acid (“JA”) overa four-day period were measured. As demonstrated in FIGS. 7 and 8, therewas a considerable decrease in total cell number among T-47D andCRL-2632 cells, respectively, treated with 12-oxo-PDA compared to thosetreated with JA. In fact, treatment with 12-oxo-PDA resulted in adecrease in total cell count. Conversely, cells treated with JA,although showing some growth inhibition relative to an untreatedcontrol, nonetheless had a net increase in total tumor cell numberduring the treatment period.

Example 7 Cyclin D1 Expression is Inhibited by 12-oxo-PDA (I)

[0143] Treatment of T-47D metastatic human breast cancer cells with12-oxo-PDA (20 μM) results in inhibition of cyclin D1 expression and ofthe hyperphosphorylation of retinoblastoma tumor suppressor protein(“Rb”). Hyperphosphorylation of Rb is a critical process and componentfor regulating the cell cycle. The phosphorylation of Rb enables cellsto pass through particular cell cycle checkpoints and to enter the Sphase of cell cycle. FIG. 9 demonstrates that treatment of T-47D cellswith 12-oxo-PDA dramatically inhibited Rb phosphorylation at the 807 and811 serine residues (it is known that the 807 and 811 serine residues ofRb are principally phosphorylated by the cyclin D1-CDK4 complex) asdetermined on Western blot analysis using a phospho-specific Rbantibody. FIG. 9 further demonstrates that the inhibition of Rbphosphorylation correlates with decreased cyclin D1 expression. Despitethese inhibitory effects, total Rb remained unchanged. Similarly,expression of β-tubulin and phosphorylation of p38 MAPK were unaffected.

Example 8 Measurement of Regulatory/Inhibitory Effects of 12-oxo-PDA (I)

[0144] T-47D cells were exposed to 12-oxo-PDA in an effort to determinethe effect of 12-oxo-PDA on gene expression. FIGS. 10, 11 and 12 show12-OPDA mediated regulation of cyclin D1, p27 and c-fos mRNA levels,respectively, in T-47D cells compared to ethanol treatment alone. Asdemonstrated in FIG. 10, treatment with 12-oxo-PDA specifically downregulates the expression of cyclin D1 mRNA, but has no significanteffect on expression of p27 (FIG. 11) and C-FOS m RNA (FIG. 12) in theT-47D cells. In fact, a greater than twenty-fold decrease in cyclin D1mRNA levels was observed in cells treated with 12-oxo-PDA relative tountreated T-47D cells. Conversely, no significant changes were observedin the p27 and C-FOS mRNA levels upon 12-oxo-PDA treatment. Thus, itappears that 12-oxo-PDA has a direct regulatory/inhibitory effect on thecyclin D1 and Rb protein. Ubiquitine expression in ethanol and 12-OPDAtreated cells has been used as internal control in each amplificationexperiment (left curves).

Example 9 Apoptosis Induced by 12-oxo-PDA (I)

[0145] Regulated cell apoptosis is essential for the development andmaintenance of the immune system. The highly regulated anti-apoptoticMCL-1 protein appears to enhance short-term survival and functions ingenotoxic cell death. Removal of MCL-1 has been shown to cause aprofound reduction in B and T lymphocyte cells in mice. Thus, deletionof MCL-1 during early lymphocyte differentiation increases apoptosis andarrests the development of B and T-lymphocytes. In this regard, onehallmark of apoptosis is activation of the caspases (e.g., caspase-3,caspase-6, and caspase-7). Activation of caspases leads to the cleavageof a 11 8-kDa PARP protein into an 89-kDa fragment.

[0146] In FIG. 13, CRL-2632 diffuse large B-cell lymphoma cells weretreated with 12-oxo-PDA to demonstrate that 12-oxo-PDA induces apoptosisand inhibits MCL-1 expression. Cells were incubated in the presence of12-oxo-PDA (20 μM) for between one and six hours. The cells were thenwashed and lysed to extract total proteins. Thereafter, 30 μg extractswere resolved on 8% SDS-PAGE gel and probed with anti-PARP, anti-MCL-1,anti-bcl-2, anti-bax and anti-bak antibodies. As shown in FIG. 12,immunoblot analysis of the extracts from cells treated with 12-oxo-PDAshowed a time-dependent cleavage of PARP (whereas untreated cells showedno PARP cleavage). Similarly, FIG. 13 illustrates the effects of12-oxo-PDA treatment on apoptosis related proteins (i.e., Bcl-2, MCL-1,Bax and Bak proteins). Specifically, 12-oxo-PDA inhibited the expressionof MCL-1, but had no effect on the expression of Bcl-2, Bax and/or Bakproteins in the CRL-2632 cells.

[0147] Synthetic coronatine (“CRNT”) can inhibit growth of CRL-2632cells (FIG. 14) and also down regulate the expression of the MCL-1protein and induce apoptosis (FIG. 15). CRL-2632 diffuse large B-celllymphoma cells (and Burkitt's lymphoma cells (“Raji”)) were treated with12-oxo-PDA and a synthetic coronatine analog (0.5 mM). Both 12-oxo-PDAand synthetic coronatine arrest growth and induce apoptosis in theCRL-2632 and Raji cells, as demonstrated in FIG. 15. Furthermore, both12-oxo-PDA and the synthetic coronatine down-regulated expression ofMCL-1 in the treated lymphoma cells. As such, the foregoing data furthersuggests that both 12-oxo-PDA and coronatine induce apoptosis inhigh-grade lymphoma cells and inhibit MCL-1 protein expression byactivation of caspase pathways.

Example 10 Induction of Tubulin Polymerization

[0148] Mitotic spindles are critical elements in a variety offundamental cellular functions including, for example, chemotaxis,membrane and intracellular scaffolding, transport, secretory processes,regulatory of cellular motility and cell division. Microtubules playessential roles in the formation, operation and regulation of themitotic spindles. As such, disruption of the regulatory functioning andapparatus of the microtubules can induce cell-cycle arrest during the Mphase of cellular development and trigger signals to induce programmedcell death. In this regard, microtubule inhibitors interfere with thetubulin dynamics of tubulin polymerization and depolymerization andresult in the inhibition of cell division. For example, anti-mitoticdrugs (e.g., taxanes and Vinca alkaloids) have been used to treatvarious kinds of human cancers. The taxanes, including paclitaxel anddocetaxel, stabilize microtubules and induce net microtubulepolymerization and are effective in the treatment of breast, lung,ovarian, bladder, head and neck cancers. The Vinca alkaloids, such asvincristine, vinblastine and vinorelbine, prevent normal polymerizationof microtubules and are important in treating leukemia, lymphoma, smallcell lung cancer and other malignancies.

[0149] In FIG. 16 it is demonstrated that 12-oxo-PDA induces tubulinpolymerization in CRL-2632 cells. In particular, it was observed thattreatment of exponentially growing CRL-2632 cells with 12-oxo-PDA (20μM) for between one and six hours resulted in the loss of tubulin insoluble but accumulation in insoluble fractions (free tubulin andmicrotubule fractions (pellet) were isolated and analyzed by Westernblot). The increase in insoluble tubulin fractions indicatespolymerization of tubulin in treated cells. From this data it appearsthat 12-oxo-PDA may interfere with tubulin polymerization anddepolymerization processes and thus inhibit cancer cell division.

[0150] In view of the foregoing results, it should be noted that theabove-treated cells each attribute their malignant behavior to differenttransformation processes and mechanisms. These varying processes includeaberrant ras oncogene activity in CRL-5985 human lung adenocarcinomacells, a non-functional p53 pathway in T-47D breast ductal carcinomacells and the excessive expression of bcl-2 anti-apoptotic factor inCRL-2632 human diffuse large B-cell lymphoma cells. Despite thesevarious mechanisms, 12-oxo-PDA and its related compounds nonethelesshave an inhibitory effect on cell growth. Thus, it is possible that theinhibitory effects of 12-oxo-PDA and its related compounds may beindependent of the particular mechanism involved in the malignanttransformation of cells. As such, 12-oxo-PDA and its related compoundsmay be particularly useful for treating malignant cells that have shownresistance to other known chemotherapeutic agents (and especiallyresistances to cell cycle and apoptosis regulatory protein agents suchas those affecting p53 and/or bcl-2).

[0151] The foregoing description is considered as illustrative only ofthe principles of the invention. Further, since numerous modificationsand changes will readily occur to those skilled in the art, it is notdesired to limit the invention to the exact construction and processshown as described above. Accordingly, all suitable modifications andequivalents may be resorted to falling within the scope of the inventionas defined by the claims that follow. The words “comprise,”“comprising,” “include,” “including,” and “includes” when used in thisspecification and in the following claims are intended to specify thepresence of stated features, integers, components, or steps, but they donot preclude the presence or addition of one or more other features,integers, components, steps, or groups thereof.

What is claimed is:
 1. A method of controlling proliferative cells in a subject, comprising administering a therapeutically effective amount of at least one compound having the formula:

wherein —is an optional double bond; A¹ and A² are independently H, Z_(m)-OR⁶, oxo, halo, Z_(m)-CN, Z_(m)-NO₂, azido, Z_(m)-NR⁶R⁷, Z_(m)-COOR⁶, Z_(m)-CONR⁶R⁷, Z_(m)-C(═O)R⁶, Z_(m)-OC(═O)R⁶, alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy, thiol, thioalkyl, Z_(m)-cycloalkyl wherein said cycloalkyl is saturated or partially unsaturated, Z_(m)-heterocycloalkyl wherein said heterocycloalkyl is saturated or partially unsaturated, or Z_(m)-Ar, wherein said alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy, Z_(m)-cycloalkyl, Z_(m)-heterocycloalkyl, and Z_(m)-Ar may be substituted or unsubstituted; A³ and A⁴ are independently alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, alkoxy, heteroalkoxy, Z_(m)-cycloalkyl wherein said cycloalkyl is saturated or partially unsaturated, Z_(m)-heterocycloalkyl wherein said heterocycloalkyl is saturated or partially unsaturated, Z_(m)-Ar, Z_(m)-O—R⁶, Z_(m)-SR⁶, Z_(m)-NR⁶R⁷, Z_(m)-C(═O)R⁶, Z_(m)-OC(═O)R⁶, Z_(m)-C(═O)OR⁶, Z_(m)-(C═O)NR⁶R⁷, or Z_(m)-NHC(═O)R⁶, wherein said alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy, Z_(m)-cycloalkyl, Z_(m)-heterocycloalkyl, and Z_(m)-Ar may be substituted or unsubstituted and wherein at least one of A³ or A⁴ is at least three atoms in length; or A³ and A⁴ together with the atoms to which they are both attached form a substituted or unsubstituted saturated or partially unsaturated ring or a substituted or unsubstituted aromatic ring having at least five atoms, wherein one or more of the atoms is optionally a heteroatom; R⁶ and R⁷ are independently H, Z_(m)-OR⁶, alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy, Z_(m)-cycloalkyl wherein said cycloalkyl is saturated or partially unsaturated, Z_(m)-heterocycloalkyl wherein said heterocycloalkyl is saturated or partially unsaturated, or Z_(m)-Ar, wherein said alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy, Z_(m)-cycloalkyl, Z_(m)-heterocycloalkyl, and Z_(m)-Ar may be substituted or unsubstituted; X is OR⁶, oxo, heteroalkoxy, O-glucosyl, thiol, thioalkyl, NR⁶R⁷, halo, CN, NO₂, or azido; Ar is aryl or heteroaryl; Z is CH₂; and m is an integer between 0 and
 10. 2. The method of claim 1, wherein A³ and A⁴ are independently

wherein n is 3, 4, 5, 6, 7, 8, 9, or 10; D₁, D₂ and D₃ are independently H, Z_(m)-OR⁶, Z_(m)-O-glucosyl, heteroalkoxy, thiol, thioalkyl, Z_(m)-NR⁶R⁷, halo, Z_(m)-CN, Z_(m)-NO₂, or azido; D₄ is H, Z_(m)-OR⁶, O-glucosyl, imino, halo, Z_(m)-CN, Z_(m)-NO₂, azido, Z_(m)-C(═O)H, Z_(m)-NR⁶R⁷, Z_(m)-COOR⁶, Z_(m)CONR⁶R⁷, Z_(m)-C(═O)R⁶, Z_(m)-OC(═O)R⁶, alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy, thiol, thioalkyl, Z_(m)-cycloalkyl wherein said cycloalkyl is saturated or partially unsaturated, Z_(m)-heterocycloalkyl wherein said heterocycloalkyl is saturated or partially unsaturated, or Z_(m)-Ar¹, wherein said alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy, Z_(m)-cycloalkyl, Z_(m)-heterocycloalkyl, and Z_(m)-Ar¹ may be substituted or unsubstituted; or D₄ and X, or D₄ and D₃ together form a lactone; and m is an integer between 0 and
 10. 3. The method of claim 1, wherein A³ and A⁴ are independently


4. The method of claim 1, wherein the compound is


5. The method of claim 1, wherein A³ and A⁴ together form a six-member ring.
 6. The method of claim 5, wherein said six-member ring contains at least one carbon-carbon multiple bond.
 7. The method of claim 5, wherein said six-member ring is aromatic.
 8. The method of claim 5, wherein said six-member ring contains at least one additional substituent group.
 9. The method of claim 8, wherein said at least one additional substituent group is selected from the group of H, OR⁶, oxo, halo, CN, NO₂, azido, NR⁶R⁷, COOR⁶, CONR⁶R⁷, C(═O)R⁶, OC(═O)R⁶, alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy, thiol, thioalkyl, Z_(m)-cycloalkyl wherein said cycloalkyl is saturated or partially unsaturated, Z_(m)-heterocycloalkyl wherein said heterocycloalkyl is saturated or partially unsaturated, or Z_(m)-Ar, wherein said alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy, Z_(m)-cycloalkyl, Z_(m)-heterocycloalkyl, and Z_(m)-Ar may be substituted or unsubstituted.
 10. The method of claim 1, wherein the compound is

wherein R¹ is

R², R³, R⁴ and R⁵ are independently H, Z_(m)-OR⁶, alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy, Z_(m)-NR⁶R⁷, Z_(m)-COOR⁶, Z_(m)-CONR⁶R⁷, Z_(m)-C(═O)R⁶, Z_(m)-OC(═O)R⁶, Z_(m)-cycloalkyl wherein said cycloalkyl is saturated or partially unsaturated, Z_(m)-heterocycloalkyl wherein said heterocycloalkyl is saturated or partially unsaturated, or Z_(m)-Ar, wherein said alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy, Z_(m)-cycloalkyl, Z_(n)-heterocycloalkyl, and Z_(m)-Ar may be substituted or unsubstituted, or R³ and R⁴ together with the atoms to which they are both attached form a saturated or partially unsaturated ring, wherein said saturated ring or partially unsaturated ring may be substituted or unsubstituted; and Y¹, Y², and Y³ are independently H, Z_(m)-OR⁶, alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy, Z_(m)-NR⁶R⁷, Z_(m)-COOR⁶, Z_(m)-CONR⁶R⁷, Z_(m)-C(═O)R⁶, Z_(m)-OC(═O)R⁶, Z_(m)-cycloalkyl wherein said cycloalkyl is saturated or partially unsaturated, Z_(m)-heterocycloalkyl wherein said heterocycloalkyl is saturated or partially unsaturated, or Z_(m)-Ar, wherein said alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy, Z_(m)-cycloalkyl, Z_(n)-heterocycloalkyl, and Z_(m)-Ar may be substituted or unsubstituted.
 11. The method of claim 10, wherein R¹ is a substituted or unsubstituted natural or unnatural amino acid.
 12. The method of claim 11, wherein R¹ is alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.
 13. The method of claim 11, wherein R¹ is 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvaline, beta-alanine, gamma-aminobutyric acid, cirtulline, homocysteine, homoserine, omithine and methionine sulfone.
 14. The method of claim 10, wherein the compound is


15. The method of claim 14, wherein said compound is


16. The method of claim 10, wherein said compound is


17. The method of claim 16, wherein said compound is


18. The method of claim 1, wherein said subject has cancer.
 19. The method of claim 1, wherein said cancer is ovarian cancer.
 20. The method of claim 1, wherein said cancer is breast cancer.
 21. The method of claim 1, wherein said cancer is lung cancer.
 22. The method of claim 1, wherein said cancer is lymphoma.
 23. The method of claim 1, wherein said method of treatment further comprises at least one of an hourly administration, a daily administration, a weekly administration, or a monthly administration of said at least one composition.
 24. The method of claim 1, wherein said administration comprises oral administration of said at least one composition.
 25. The method of claim 1, wherein said administration comprises injection of said at least one composition.
 26. The method of claim 1, wherein said administration comprises intravenous administration of said at least one composition.
 27. The method of claim 1, wherein said subject is an animal.
 28. The method of claim 1, wherein said subject is a human.
 29. A method for controlling proliferative cells in a subject, comprising supplying to said subject at least one compound of the formula:


30. A method for controlling proliferative cells in a subject, comprising supplying to said subject a compound of the formula:


31. A method for controlling proliferative cells in a subject, comprising supplying to said subject a compound of the formula:


32. A method for conducting a clinical trial comprising supplying to a subject at least one compound of the formula:

wherein said composition contains at least one additional carbon-carbon multiple bond; and wherein one or both of R¹ and R² define a structure selected from the group consisting of (a) at least one substituent selected from the group of hydrogen, alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, alkoxy, heteroalkoxy and (b) a second ring structure of at least five atoms.
 33. The method of claim 1, wherein A⁴ is

n is 3,4, 5, 6, 7, 8, 9, or 10; and D₄ is H, Z_(m)-OR⁶, O-glucosyl, imino, halo, Z_(m)-CN, Z_(m)-NO₂, azido, Z_(m)-C(═O)H, Z_(m)-NR⁶R⁷, Z_(m)-COOR⁶, Z_(m)-CONR⁶R⁷, Z_(m)-C(═O)R⁶, Z_(m)-OC(═O)R⁶, alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy, thiol, thioalkyl, Z_(m)-cycloalkyl wherein said cycloalkyl is saturated or partially unsaturated, Z_(m)-heterocycloalkyl wherein said heterocycloalkyl is saturated or partially unsaturated, or Z_(m)-Ar¹, wherein said alkyl, allyl, alkenyl, alkynyl, heteroalkyl, heteroallyl, heteroalkenyl, heteroalkynyl, heteroalkoxy, Z_(m)-cycloalkyl, Z_(m)-heterocycloalkyl, and Z_(m)-Ar¹ may be substituted or unsubstituted.
 34. A method of controlling proliferative cells in a subject, comprising administering a therapeutically effective amount of at least one compound having the formula:


35. The method of claim 34, wherein R¹ is alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.
 36. The method of claim 34, wherein R¹ is 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvaline, beta-alanine, gamma-aminobutyric acid, cirtulline, homocysteine, homoserine, omithine and methionine sulfone.
 37. A pharmaceutical composition for controlling proliferative cells in a subject, comprising a therapeutically effective amount of a compound having the formula:

and a pharmaceutically acceptable carrier.
 38. A pharmaceutical composition for controlling proliferative cells in a subject, comprising a therapeutically effective amount of a compound having the formula:

and a pharmaceutically acceptable carrier.
 39. A pharmaceutical composition for controlling proliferative cells in a subject, comprising a therapeutically effective amount of a compound having the formula:

and a pharmaceutically acceptable carrier. 